Oils of marine origin are naturally rich sources of polyunsaturated fatty acids. The most beneficial polyunsaturated fatty acids particularly have the following formulas:

These fatty acids, although they are necessary for the body to function correctly, are not synthesised naturally by the human body. Therefore, the intake thereof is associated with a daily intake via diet. Dietary sources of polyunsaturated fatty acids are vegetable oils (essentially ω-6 and ω-9 type fatty acids) and fish oils which particularly contain large quantities of ω-3 type fatty acids. The latter are very well known for their beneficial effects on health (cardiovascular diseases, auto-immune diseases, inflammations, etc.). Polyunsaturated fatty acids are classified according to the position of the first double bond, from the terminal methyl function. In this way, in the nomenclature ω-x or n-x, the x corresponds to the position of said first unsaturation. The majority of polyunsaturated fatty acids of biological interest belong to the ω-6 (arachidonic acid) or ω-3 (EPA, DHA) family. In addition, in the nomenclature, the number of carbons forming the chain is also defined; in this way, EPA is described as C20:5 and DHA as C22:6. The numbers 5 and 6 correspond to the number of carbon chain unsaturations displayed by EPA and DHA, respectively. Fish oils are essentially used to isolate and concentrate ω-3 type fatty acids.
Conventional fish oil enrichment methods (article by V. K. Mishra et al., Food Research International, 1993, 26, 217-226) are based on selectivity with respect to the length of the fatty acid chains forming the oils or their degree of unsaturation. The most commonly used enrichment processes are performed on fatty acids or the corresponding esters by means of:                Crystallisation        Countercurrent extraction        Molecular distillation        Absorption chromatography.        
Most of the time, different processes are combined with a view to obtaining a high level of enrichment. In addition, these processes involve the following drawbacks:
Processes performed at high temperatures (distillation) give rise to multiple thermal degradation products of fatty acids (isomerisation, peroxidation, oligomerisation). For this reason, it is recommended to work at low temperatures, advantageously at temperatures below 100° C.
The drawback of chromatography techniques is based on the use of massive quantities of solvents, which are frequently toxic. In addition, large-scale production based on such techniques is far from easy.
For these reasons, alternative methods have been developed. They are based on the use of supercritical fluids:                Supercritical CO2 fractionating process        Supercritical chromatography.        
The fractionating process of fatty acid ethyl esters by means of supercritical CO2 has already been described extensively in the literature. However, it should be noted that the majority of the processes cited describe ω-3 or eicosapentaenoic acid (EPA), and not DHA, enrichment.
One of the operating parameters of the supercritical CO2 fractionating process is the level of supercritical CO2. This level is defined by the ratio of the CO2 flow over the flow of injected fatty acid solution. In this way, at high levels of supercritical CO2, the selectivity is increased to the detriment of the yield. At low levels of supercritical CO2, the yield is favoured while the selectivity is decreased.
In this way, Nilsson et al. (JAOCS 1988, 65 (1), 109-117) describe a batch process making it possible to obtain several fractions (0.1 to 0.2 g) rich in EPA and DHA respectively. For this purpose, the authors worked at pressures between 220*105 Pa and 250*105 Pa. In addition, they produced a temperature gradient in the column to generate an internal reflux (from 20° C. at column bottom to 100° C. at column head). The levels of supercritical CO2 defined are very high, of the order of 100 to 500.
Some tests make it possible to obtain, from ethyl esters pre-treated with urea, fractions having a DHA content of the order of 90%, but with levels of supercritical CO2 of 500.
Using non-treated ethyl esters, the authors described DHA-rich fractions (content between 53 and 60%) but for levels of supercritical CO2 of the order of 300 to 400.
Therefore, this process has the following drawbacks: it consists of a batch process. The level of supercritical CO2 used is too high and the process must therefore have a low yield, which cannot be used industrially. In fact, this induces additional energy costs and therefore lower productivity. The temperature of 100° C. at the column head may induce fatty acid degradation. However, this is the temperature recommended by the authors. The pressures used are too high and, if they were reduced, there would be an increase in the level of supercritical CO2. Moreover, this article recommends the use of a temperature gradient within the column and, in particular, an internal reflux in order to improve separation and therefore enrichment. However, the presence of such a reflux decreases the productivity of the process.
Kado et al. (JP2005-255971) describe in their patent a fish oil ethyl ester enrichment process with EPA and DHA. The temperature and pressure ranges claimed are 35-200° C. and 100*105 Pa-500*105 Pa. The authors recommend two successive extractions in order to obtain high contents. A first extraction is performed on the raw material, and a second extraction is performed on the residue from the first operation. The column used is 3 m high for an inner diameter of 50 mm. It comprises 6 separate heating chambers. The two successive extractions complicate the process and render it industrially inapplicable.
The levels of supercritical CO2 used to obtain high DHA contents are high (of the order of 127). Therefore, the DHA yield is low. Moreover, the authors recommend the application of a temperature gradient (which makes it possible to obtain a rectification effect) in the column. Therefore, the productivity is restricted.
Lucien et al. (Australasian biotechnology, 1993, 3 (3), 143-147) describe a process to obtain EPA and DHA ethyl ester-enriched extracts from 17/12 (EPA/DHA) sardine oil. An internal reflux generated by a heating chamber placed at the column head makes it possible to improve the contents obtained: EPA and DHA contents of 42% and 54%, respectively, may be achieved. The best operating conditions in this configuration are 150*105 Pa, 40° C. in the column and a reflux temperature of 100° C. The level of supercritical CO2 used is not specified.
The table below contains the results obtained:
ExtractionRefluxPressuretemperaturetemperature[EPA][DHA](Pa)(° C.)(° C.)(%)(%)150 * 10540602134150 * 10540803339150 * 105401004254
Therefore, this process has the following drawbacks: the temperature of 100° C. in the column head may induce fatty acid degradation. However, this is the temperature recommended by the authors. Moreover, this article recommends the use of a temperature gradient within the column and, in particular, an internal reflux in order to improve separation and therefore enrichment. However, the presence of such a reflux decreases the productivity of the process.
Finally, Zhu et al. (Proceedings of the 3rd International Symposium on supercritical fluids—Strasbourg, 1994) describe a fatty acid ethyl ester extraction/fractionating process in order to obtain fractions rich in EPA and DHA, respectively. For this purpose, a CO2 flow extracts the fatty acid esters contained in an extractor, and this CO2 flow charged with esters is then fractionated in a column. A temperature gradient and pressure programming are performed in the column, in order to improve the selectivity with respect to the compounds under test. This batch process makes it possible to isolate a fraction representing 12% of the DHA used with more than 50% purity.
Therefore, this process has the following drawbacks: it consists of a batch process and, for this reason, it may comprise pressure programming. The level of supercritical CO2 used is very high (211) and the process must therefore have a low yield, which is not industrially usable. In fact, this induces additional energy costs and therefore lower productivity. Moreover, this article recommends the use of a temperature gradient within the column and, in particular, an internal reflux in order to improve separation and therefore enrichment. However, the presence of such a reflux decreases the productivity of the process.
Therefore, the majority of the processes described in the literature use a high level of supercritical CO2, which gives a very low DHA yield and therefore a process which is not industrially feasible. In addition, for a process to be industrially feasible, it is also necessary not to work at an excessively high pressure. However, when the pressure decreases, the density decreases and it is therefore necessary to increase the level of supercritical CO2 to obtain an equivalent DHA content. Therefore, it is necessary to find a compromise between the pressure, level of supercritical CO2 and yield, in order to obtain a beneficial DHA enrichment, i.e. at least 50%. Moreover, only continuous processes are of interest from an industrial point of view.
Finally, in order to prevent the degradation of fatty acids and of DHA in particular, it is advisable to work at a low temperature, i.e. a temperature less than 100° C. and in particular less than or equal to 70° C.
In addition, it is also advisable to find a process making it possible to enrich a fatty acid solution with DHA only using a single countercurrent fractionating step, not requiring pre-treatment of the fatty acids with urea.